The ability to acquire and analyse DNA sequence data has increased phenomenally over the past few years. As a result, nucleic acid analysis has become increasingly important in many areas of biology, biotechnology and medicine.
New sequencing technologies such as those based on sequencing by synthesis have the ability to produce raw sequence data at a rate and quantity many orders of magnitude higher than previously possible with Sanger sequencing and other conventional methods. However, there are a number of important differences in the sequence data that is produced. Whilst the introduction of new sequencing technologies has led to a significant increase in the amount of raw nucleic acid sequence obtained, there has also been a concomitant reduction in read length. Despite this, in terms of sequence assembly it is now possible to produce high depth sequence data for a medium sized genome from just a single sequencing run.
The starting point for many nucleic acid analyses is genomic DNA which may contain tens of millions of base pairs. Therefore, fragmentation of the nucleic acid sequence is generally required to reduce the size of the sequence into smaller parts that are more amenable to manipulation.
Fragmentation of nucleic acids is generally performed by enzymatic, chemical or mechanical means. A primary disadvantage of each of these methods is that although the nucleic acid may be randomly fragmented, the resulting fragments are distributed across a wide range of sizes. As a result, further purification steps such as gel purification are required to select fragments of suitable size for a particular application. Since new sequencing technologies generally provide shorter read lengths, the use of larger fragments is less efficient both in terms of sequence coverage and utilisation of material, for example. Size selection based on electrophoresis and gel excision of the desired size range leaves the bulk of the starting nucleic acid in the electrophoresis gel.
There is a need for sample preparation methods where the sample is treated to obtain material of a desired length, and all of the sample is available for subsequent use, especially in cases where the amount of material is limited, such as biopsies, laser captured cells, limited archival tissues, embryoid bodies, small model systems, and difficult to cultivate organisms such as Microsporidia. The present invention satisfies this need and provides other advantages as well.